Brucella bacteria are widely distributed throughout the world causing huge losses in agriculture and threatening human health through zoonotic infection. In this article, a label-free amperometric immunosensor for the quantitative detection of a low-concentration Brucella-positive standard serum was developed to identify diseased livestock earlier and thereby reduce economic losses. The developed immunosensor consists of a screen-printed gold electrode (SPGE) and a signal-acquiring interface. The SPGE was first functionalized with cysteamine for the immobilization of Brucella melitensis. Then, Brucella-positive standard sera of various concentrations were introduced dropwise onto the surface of the electrode and captured by B. melitensis. This resulted in current changes that could be measured with differential pulse and square wave voltammetries in the electrolyte solution using an electrochemical workstation. There was a linear relationship between the variation in peak current and the logarithmic value of Brucella-positive standard serum concentrations from 1 × 10−5 to 1 × 102 IU/mL. Furthermore, the second-derivative transformation was applied, and the optimized linear correlation between the variation in peak value and the corresponding Brucella-positive standard serum concentration was established. Moreover, the regression equation has a lower detection limit, a higher goodness of fit, and a smaller residual sum of squares.